Dysregulation of endothelial cell mediated anti-inflammatory pathways can lead to cardiovascular disease, the leading killer in the western world, and severe sepsis, which accounts for 220,000 deaths/year in the United States. Our long term goal is to elucidate novel anti-inflammatory processes of endothelial cells, which may lead to new approaches to combat severe sepsis, heart attack and stroke. Antithrombin (AT) is a blood protein that, in part, prevents inflammation through the existence of multiple isoforms (beta-AT, alpha-AT, L- AT);however, the anti-inflammatory mechanisms are poorly understood. beta-AT preferentially partitions to the blood vessel wall and is considered to exhibit high anti-inflammatory activity. Thus, partitioning of specific AT forms may convey a strong anti-inflammatory tone to the blood vessel wall. Partitioning may also serve to enhance the stability of anti-inflammatory AT isoforms, for AT forms that lack partitioning are extremely short-lived. AT biology is thought to be regulated by an endothelial cells component called HSAT+. However, it is unclear whether HSAT+ is involved in AT partitioning and anti-inflammatory activity. We have determined that HSAT+ is required for endothelial cell internalization of AT and seek to evaluate the involvement of HSAT+ and AT endocytosis in AT partitioning and AT anti-inflammatory activity. We propose to: (1) Evaluate the role of HSAT+ in the anti-inflammatory activity of AT. This will be accomplished by studying the efficacy of AT therapy in treating septic shock induced in HSAT+ deficient or wild-type mice. (2) Elucidate molecular mechanisms by which HSAT+ mediates AT internalization. Cell biologic experiments will be conducted to determine if AT anti-inflammatory signaling is associated with a unique pathway of AT internalization. (3) Establish the role of HSAT+ in regulating the partitioning of distinct AT isoforms. To examine if HSAT+ preferentially influences the vascular partitioning and longevity of AT forms with high anti-inflammatory activity, we shall use novel molecular imaging techniques to follow the partitioning and fate of AT isoforms injected into HSAT+ deficient or wild-type mice. Together, these Aims will demonstrate novel roles for HSAT+ and provide foundations for elucidating the involvement of HSAT+ mediated AT partitioning in protecting against inflammatory processes of the cardiovascular system. Lay description: Vascular inflammation is a common denominator of numerous major diseases, such as heart attack and stroke, which are the leading killers in the western world. The body normally inhibits vascular inflammation and we wish to discover molecules involved in this processes. Such a discovery could lead to the development of novel therapeutics to combat the devastating consequences of vascular inflammation.